Evaluation of Antioxidant Activity of Vegetables from Okinawa Prefecture and Determination of Some Antioxidative Compounds

Transcription

1 Food Sci. Technol. Res., +, (+), 2 +.,,**0 Evaluation of Antioxidant Activity of Vegetables from Okinawa Prefecture and Determination of Some Antioxidative Compounds Gouki MAEDA +, 0, Kensaku TAKARA,, Koji WADA,, Tomoyuki OKI -,MamiMASUDA -, Toshio ICHIBA., Yoshihiro CHUDA /, Hiroshi ONO / and Ikuo SUDA - + Okinawa Prefectural Agricultural Experiment Station,.,,, Sakiyama-cho, Naha, Okinawa 3*- *2+., Japan, Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, + Senbaru, Nishihara-cho, Okinawa 3*- *,+-, Japan - National Agricultural Research Center for Kyushu Okinawa Region, National Agricultural Research Organization,,.,+ Suya, Nishigoshi, Kikuchi, Kumamoto ,, Japan. Okinawa Industrial Technology Center, +,, Suzaki, Uruma-shi, Okinawa 3*.,,-., Japan / National Food Research Institute,, +, Kan-nondai, Tsukuba-shi, Ibaraki -*/ 20.,, Japan 0 Present address: Okinawa Industrial Technology Center, +,, Suzaki, Uruma-shi, Okinawa 3*.,,-., Japan Received June 3,,**/; Accepted January +,,,**0 We examined the phenolic contents of./ species of vegetables and their radical scavenging activities for +,+-diphenyl-,-picrylhydrazyl radical (DPPH ). High contents of phenolic compounds and strong DPPH scavenging activities were observed in extracts from Nishiyomogi, Ryukyuyomogi, Aojiso, Moroheiya, Hosobawadan, Bajiru, Ensai, Yaeyamakazura, Suizenjina, Botanbouhuu and Uikyou. Eight of these vegetables are traditional foods in Okinawa. These showed strong radical scavenging activities for the superoxide radical (O, )andtert-butylhydroperoxyl radical (t-buoo ). We found five antioxidative phenolic compounds, D ( )-chicoric acid, chlorogenic acid, ca#eic acid, luteolin 1-O-b-D-glucopyranoside, and luteolin 1-O-b-D-glucuronide, in Hosobawadan (Crepidiastrum lanceolatum Nakai). Their detailed structures were determined using + H-NMR, +- C-NMR, MS, and UV spectrometry. Keywords : antioxidant, polyphenol, luteolin, chicoric acid, Crepidiastrum lanceolatum Introduction The traditional food culture of Okinawa is rich in seaweed, greenish-yellow vegetables, medicinal plants, and fat, and low in salt, compared to those of other regions in Japan. It is noted that Okinawa is a high-fat-ingestion area due to high consumption of pork; however, the Okinawa population has a low death rate from circulatory organ system diseases resulting from arteriosclerosis, such as cardiopathy and cerebral hemorrhages (Syuyama,,***). A similar phenomenon has occurred in France, with a low incidence of coronary heart disease and arteriosclerosis despite a high-fat diet, which is famous as the French paradox (Renaud and Lorgeril, +33,). This paradox is reported to be related to the high prevalence of red wine consumption; the phenolic compounds contained in red wine are thought to be responsible for anti-arteriosclerosis action (Frankel et al., +33-; Frankel et al., +33/; Kondo et al., +33.; Kerry and Abbey, +331; Zouet Abbreviations : DPPH, +,+-diphenyl-,-picrylhydrazyl; BHA, butylated hydroxyanisole; SOD, superoxide dismutase; t-buoon tert-butylhydroperoxyl radical; FAB-MS, fast atom bombardment spectrometry ; APCI-MS, atmospheric pressure chemical ionization mass spectrometry. * To whom correspondence should be addressed. koji wada@agr.u-ryukyu.ac.jp al., +333). Phenolic compounds with antioxidant activities are expected to be present in vegetables eaten daily in Okinawa. In this study, we measured the phenolic contents of vegetables eaten in Okinawa, especially green vegetables and fruit-vegetables, and evaluated their radical scavenging or antioxidant activities using several assay systems, which included assays for evaluating +,+-diphenyl-,- picrylhydrazyl radical (DPPH ) scavenging activity, superoxide radical (O, ) scavenging activity, tertbutylhydroperoxyl radical (t-buoo ) scavenging activity, and b-carotene-fading activity. HPLC analysis was also performed for the purpose of identifying several of the antioxidants. Hosobawadan is used as an edible plant and as a folk medicine for colds, diarrhea, arteriosclerosis, and hypertension and for treatment of gastrointestinal diseases in Okinawa, where it is known by the local name of Nigana or Njana. We also isolated and identified several antioxidants from Hosobawadan, and evaluated their antioxidant activities. Materials and Methods Materials Forty-four di#erent vegetables were purchased from retail stores; the hihatsumodoki leaves were

2 Antioxidant Activity of Vegetables from Okinawa Prefecture 9 donated. The edible parts of the vegetables were lyophilized and then powdered using a mill and stored at,* until used. The vegetable powders were used in this study as indicated in Table +. Preparation of vegetable extracts Vegetable powder was extracted with 2* EtOH at a /-fold volume of fresh weight at 2* for -* min. The extraction was centrifuged at -*** rpm for +* min and the supernatant was used. Determination of phenolic content The total phenolic content of the vegetable extract was measured using the Folin-Denis assay (Tateyama et al., +331). Briefly, the reaction mixture, containing,.* ml of vegetable extract with,.* ml of Folin-Denis reagent and,.* ml of +* sodium carbonate, was allowed to stand for + hatroom temperature, and then measured at 1** nm using a spectrophotometer (UV+0*, Shimadzu, Japan). The total phenolic contents were expressed as mg of gallic acid equivalent/+** g of fresh weight. Measurement of DPPH scavenging activity The DPPH scavenging activity of the vegetable extract was examined according to the method of Oki et al. (,**+). Briefly, /* ml of the vegetable extract, /* ml of,* EtOH, and /* ml of*., M-morpholinoethanesulfonic acid (MES) bu#er at ph 0.* were placed in a 30-well microplate. The vegetable extract was diluted with 2* EtOH in six stages. The reaction was initiated by adding /* ml of2** mm DPPH in EtOH. After the reaction mixture was left to stand for,* min at room temperature, its absorbance at /.* nm was measured by microplate reader. DPPH scavenging activity was expressed as mmoles Trolox equivalent/+** g of sample using the standard Trolox curve. Measurement of antioxidant activity by b-carotene bleaching method Prevention of the auto-oxidation of emulsified linoleic acid in vegetable extracts was determined by a method that measures the rate of discoloration of b-carotene coupled with the oxidation of linoleic acid, as described by Tsushida et al. (+33.). That is,,* mg Table +. Vegetables and used as experimental materials, obtained from retail stores in Okinawa Prefecture.

3 10 of linoleic acid and,** mg of Tween.* were placed in a flask, and a solution of *./ mg b-carotene in chloroform was added. After removal of the chloroform using nitrogen gas, 3* ml of distilled water and +* ml of phosphate bu#er at ph 0.2 were added to the flask. Next, *.+ ml of the vegetable extract and..3 ml of linoleic acid-bcarotene solution were added to the test tube with shaking. Samples without the vegetable extract and with + mg of -(,)-t-butylated.-hydroxyl anythol (BHA) were used as the control sample and the standard substance, respectively. Samples were subjected to oxidation at /* for + h, and the absorbance values at +/ min and./ min were measured at.1* nm using a spectrophotometer. The antioxidant activity (A) was calculated using the following equation: A V./ min V +/ min B./ min B +/ min G. MAEDA et al. where V./ min absorbance at./ min with vegetable extract or control; V +/ min absorbance at +/ min with vegetable extract or control; B./ min absorbance at./ min with BHA; B +/ min absorbance at +/ min with BHA. Measurement of O, scavenging activity The O, scavenging activity of each vegetable extract was measured by the electron spin resonance (ESR)-trapping method described by Oki et al. (,**+). The reaction mixture contained /* ml of, mm hypoxanthine in a +** mm sodium phosphate bu#er (ph 1.-), -/ ml of /./ mm diethylenetriamine-n, N, N, N, N -pentaacetic acid (DTPA) in bu#er, +/ ml of3., M /,/-dimethyl-+-pyroline-noxide (DMPO), /* ml of vegetable extract in 2* EtOH and /* ml of*.. U/ml xanthine oxidase (XOD) in a bu#er. The vegetable extract was diluted with 2* EtOH in five stages. The mixture was transferred to a flat quartz cell (JEOL LC-++ cuvette) designed for the analysis of aqueous solutions, and the cell was placed in the cavity of the ESR spectrometer (JES-RE+X, JEOL Co., Ltd., Tokyo Japan). Recording of the ESR spectra started exactly 0* sec after the addition of the XOD solution under the following conditions: modulation frequency, +** khz; modulation amplitude, *.+ mt; scanning field, --/.2/ mt; receiver gain,.**; response time, *.+ sec; sweep time,,.* min; microwave power, 2 mw, and microwave frequency, 3../* GHz. Mn (II) doped in MgO was used as an internal standard. The O, scavenging activity was expressed as unit of SOD per +** g of fresh weight. Measurement of t-buoo scavenging activity The t-buoo scavenging activity of the vegetable extract was measured using the method described by Oki et al. (,**,). Briefly, +,* ml of vegetable extract and 30* ml of the mixed solution were placed in a,.-well microplate. The vegetable extract was diluted with 2* EtOH in four stages. The mixed solution consisted of +,* ml of0 mm DTPA, +,* ml of+, mm t-buooh, +,* ml of0* mm luminol, and 0** ml of phosphate-bu#ered saline at ph 1.-. The reaction was initiated by adding +,* ml of *.0 mg/ml methemoglobin. The chemiluminescence was measured by an analyzer equipped with a charge-coupled device (CCD) camera (type CLA-IMG,, Tohoku Electronic Industrial Co., Sendai, Japan). The t-buoo scavenging activity was determined based on the grade of luminescence. HPLC-photodiode array detection (PDA) analysis of Hosobawadan extract Phenolic compounds in the 2* EtOH extract from dried Hosobawadan powder were analyzed using an LC-+*AVP system coupled with an SPD-M+*AVP photodiode array detector (Shimadzu, Japan). Separation was performed on a,/*..0 mm i.d. Wakosil-II /C+2RS column (Wako, Japan) operating at.* and a flow rate of +.* ml/min. Elution was e#ected using the linear gradient from / to +** of, formic acid MeOH/H,Oin,/ min. The PDA data were recorded in the,** 2** ranges, with --* nm as the detection wavelength. -,.-O-dica#eoylquinic acid, -,/-O-dica#eoylquinic acid,.,/-o-dica#eoylquinic acid, -,.,/-O-trica#eoylquinic acid, chlorogenic acid and ca#eic acid were used as authentic standards. The compounds were identified by comparison with the retention times and UV spectra of authentic standards. Isolation of antioxidants from Hosobawadan The antioxidative compounds from Hosobawadan were isolated using the procedure of Tsushida et al. (+33.). Dried Hosobawadan powder (equivalent to + kg of fresh weight) was extracted with 2 Lof/* EtOH at 2* for -* min and then filtered. The resulting filtrate was evaporated to dryness. The residue was dissolved in distilled water and partitioned with hexane (+ L - times). The water layer was then evaporated under vacuum until dry, and dissolved in a suitable quantity of *., formic acid. The crude extract was fractionated into two fractions by column chromatography using a /*..* cm i.d., Toyo Pearl HW.*F column (Tosoh, Japan) and a linear gradient from * to +** MeOH/H,O. Detection was carried out at wavelengths of,2* and --* nm. Two fractions were subjected to preparative HPLC (column: Shimadzu Shimpack PRC-ODS,,/*,* mm i.d. (Shimadzu, Japan); elution:,* or -* MeOH) to give compounds + (-,.0 mg),, (*./ mg), - (+-*.- mg),. (++.* mg), and / (,-./ mg). + Hand +- C-NMR spectra and MS data were obtained using a JNM-.** (JEOL, Japan) and a JMS-1** (JEOL, Japan), respectively. Specific rotations were obtained using a DIP-+2* digital polarimeter (JASCO, Japan). Compound -, D-chicoric acid (di-e-ca#eoyl-(,s, -S)-( )- tartaric acid): White powder; UV (2* EtOH) l max --+ nm; ESI-MS (negative) m/z.1- [M-H] ; + H NMR (CD - OD, /** MHz) d 1.01 (,H, d, J +/.1 Hz, H-1, H-1 ), 1.++ (,H, d, J,.* Hz, H-,, H-, ), 1.*+ (,H, dd, J 2.,, +.2 Hz, H-0, H-0 ), 0.2, (,H, d, J 2.+ Hz, H-/, / ), 0.-3 (,H, d, J +/.3 Hz, H-2, 2 ), and /.20 (,H, s, H-,, H--); +- C NMR (CD - OD, +,/ MHz) d+03.. (C-+,.), +01./ (C-3, 3 ), (C-.,. ), +.2./ (C-1, 1 ), (C--, - ), +,1.- (C-+, + ), +,-.. (C-0, 0 ), (C-/, / ), ++/., (C-,,, ), ++-./ (C-2, 2 ), and 1,.- (C-,, -); [a],. D -,+ (c +.-*, MeOH) (The reference value (Lamidey et al.,,**,) is[a],, D -.* (c +.*2, MeOH)). Compound., luteolin 1-O-b-D-glucopyranoside: Yellow powder; UV (2* EtOH) l max -/* nm; APCI-MS (negative) m/z..1 [M-H] ; + H NMR (DMSO-d 0, /** MHz) d 1... (+H, dd, J 2.,,,.* Hz, H-0 ), 1..+ (+H, d, J,.* Hz, H-, ), 0.3* (+H, d, J 2., Hz, H-/ ), 0.12 (+H, d, J,.* Hz, H-2), 0.1.

4 Antioxidant Activity of Vegetables from Okinawa Prefecture 11 Table,. Polyphenol contents and antioxidant activities of vegetables from Okinawa. (+H, s, H--), 0..- (+H, d, J,.* Hz, H-0), /.*1 (+H, d, J 1./ Hz, H-+ ), and (.H, m, H-,, -,., / ); +- C NMR (DMSO-d 0, +,/ MHz) d (C-.), +0../ (C-,), +0,.3 (C-1), (C-/), +/0.3 (C-3), +/*.* (C-. ), +./.2 (C-- ), +,+.- (C-+ ), (C- 0 ), ++0.* (C-/ ), (C-, ), +*/.- (C-+*), +*-., (C--), 33.3 (C- + ), 33.3 (C-0), 3..1 (C-2), 11., (C-/ ), 10.. (C-- ), 1-.+ (C-, ), 03./ (C-. ), and 0*.0 (C-0 ). Compound /, luteolin 1-O-b-D-glucuronide: Yellow powder; UV (2* EtOH) l max -/* nm; APCI-MS (negative) m/z.0+ [M-H] ; + H NMR (DMSO-d 0, /** MHz) d 1... (+H, dd, J 2..,,.- Hz, H-0 ), 1..+ (+H, d, J,.+ Hz, H-, ), 0.3* (+H, d, J 2., Hz, H-/ ), 0.2+ (+H, d, J,.+ Hz, H-2), 0.1, (+H, s, H--), 0../ (+H, d, J,.+ Hz, H-0), /.,1 (+H, d, J 1.- Hz, H-+ ),..*- (+H, d, J 3./ Hz, H-/ ), and ,1 (-H, m, H-,, -,. ); +- C NMR (DMSO-d 0, +,/ MHz) d (C-.), (C-,), +0,./ (C-1), +0+., (C-/), +/1.+ (C-3), +/*.* (C-. ), +./.2 (C-- ), +,+.. (C-+ ), ++3., (C-0 ), ++0.* (C-/ ), +,+.. (C-, ), +*/./ (C-+*), +*-., (C--), 33.. (C-+ ), 33.. (C-0), 3..0 (C-2), 1/.. (C-/ ), 1/.1 (C-- ), 1,.2 (C-, ), 1+.- (C-. ), and 1*.- (C-0 ).

5 12 G. MAEDA et al. Fig. +. t-buoo scavenging activity of Okinawan traditional vegetables. Results and Discussion Phenolic content The phenolic content of the vegetables varied over a wide range from /.2 *.- to ,.0 mg/+** g fresh weight, and the average value was mg/+** g fresh weight (Table,). Interestingly, among eleven vegetables with values greater than the average for phenolic content, eight (Nishiyomogi, Ryukyuyomogi, Hosobawadan, Ensai, Yaeyamakazura, Botanbouhuu, Suizenjina and Uikyou) are vegetables traditionally eaten in Okinawa. DPPH scavenging activity We evaluated the DPPH scavenging activity of./ vegetable extracts. There are some reports that the DPPH scavenging activity can be correlated with phenolic content (Takahata et al.,,**+). The eight vegetables with higher phenolic contents (Nishiyomogi, Ryukyuyomogi, Hosobawadan, Ensai, Yaeyamakazura, Botanbouhuu, Suizenjina and Uikyou) showed high DPPH scavenging activity (Table,). We also confirmed in this study a high correlation (R *.3,21) between the DPPH scavenging activity of the vegetables from Okinawa and their phenolic contents. Thus, it was suggested that phenolic compounds may be contributors to DPPH scavenging activity (Table,). Antioxidant activity by b-carotene bleaching method Tsushida et al. (+33.) have evaluated the antioxidant activity of./ vegetable extracts using a method that measured the rate of discoloration of b-carotene coupled with the oxidation of linoleic acid, and reported that the activity was correlated at *.103. with its phenolic content. The eight vegetables with higher phenolic contents (Nishiyomogi, Ryukyuyomogi, Hosobawadan, Ensai, Yaeyamakazura, Botanbouhuu, Suizenjina and Uikyou) also showed high b-carotene bleaching activities, greater than + mg/+** ml BHA solution (Table,). O, scavenging activity and t-buoo scavenging activity Excess production of free radicals, such as O,, hydroxyl radical (OH ), and alkylperoxyl radical (ROO ), induces oxidative damage in cells and DNA. This damage (in vivo) is assumed to be associated with aging, cancer, arteriosclerosis, and other lifestyle-related diseases. O, is the initial active oxygen species, which immediately converts into other active oxygen radicals (OH H,O,, + O,, ROO ) in biological systems. The lipidperoxyl radical (LOO ), which possesses intermediate reactive activity and a long lifetime, is known to cause various diseases in vivo (Terao, +33*). The eight traditional vegetables with high polyphenol contents also showed high activities for O, scavenging and t-buoo scavenging (Table, and Fig. +). These results indicate that the eight vegetables traditionally eaten in Okinawa are possess both high phenolic contents and radical scavenging activity. HPLC-PDA analysis of Hosobawadan extract Among the eight vegetables, Nishiyomogi, Ryukyuyomogi, Hosobawadan and Yaeyamakazura showed very strong radical scavenging activities (Table, and Fig. +). Ichiba et al. (,***) reported the presence of -,.-O-dica#eoylquinic acid and -,/-O-dica#eoylquinic acid as major antioxidant compounds in Nishiyomogi. Furthermore, ca#eic acid, chlorogenic acid, -,.-O-dica#eoylquinic acid, -,/-O-dicaffeoylquinic acid,.,/-o-dica#eoylquinic acid, and -,.,/-Otrica#eoylquinic acid have been isolated from sweet potato leaf (Yoshimoto et al.,,**,) andconvolvulaceae as well as Yaeyamakazura. However, there have been no reports on the phenolic constituents of Hosobawadan. We attempted to isolate antioxidative compounds from Hosobawadan. The 2* EtOH extract gave chromatograms with several well-separated peaks during HPLC- PDA analysis (Fig.,). Peaks + and, were thought to be chlorogenic acid and ca#eic acid, based on their retention times and UV spectra when compared with those of authentic compounds. Peak. was a major component in the chromatogram of 2* EtOH extract from Hosobawadan (Fig.,), but did not match di- or trica#eoylquinic acid found in Nishiyomogi and sweet potato leaf. For further structural identification of peak., purification of the /* EtOH extract and several instrumental analyses were conducted. Isolation of some constituents from Hosobawadan and their antioxidative activities Five compounds were isolated from the /* EtOH extract of Hosobawadan, and their identities were determined by comparing their + H and +- C NMR, MS, and UV spectra with reference values (Morishita et al., +32.; Lamidey et al.,,**,; Veitet al., +33+; Wang et al., +332; Lu and Foo,,***). Compounds + and, were chlorogenic acid and ca#eic acid, respectively.

6 Antioxidant Activity of Vegetables from Okinawa Prefecture 13 Fig.,. HPLC chromatogram of 2* EtOH extract (A, Yaeyamakazura; B, Nishiyomogi; and C, Hosobawadan). Peak +, chlorogenic acid; peak,, ca#eic acid; peak -, isochlorogenic acid; and peaks., /, and 0, unknown. Table -. DPPH radical scavenging activity of phenolic compounds in Hosobawadan. Fig. -. Structures of compounds purified from Hosobawadan. Compound -, which was consistent with peak. from its HPLC-PDA chromatogram (Fig.,), was determined to be chicoric acid (dica#eoyltartaric acid). Chicoric acid contains a tartaric acid moiety, and an enantiomer exists. Compound - was identified as D( )-chicoric acid, because the specific rotation was [a],. D -,+ (c +.-*, MeOH) (the reference value (Lamidey et al.,,**,) is[a],, D -.* (c +.*2, MeOH)). The chicoric acid content of Hosobawadan used for this study was quantified by HPLC-PDA analysis and revealed as +3*.+ mg/+** g fresh weight, equivalent to 0 +, times the chicoric acid content of endive (the reference value (Harrmann, +323) is mg/+** g fresh weight). It has been reported that chicoric acid can inhibit HIV-+ integrase in extracellular enzyme preparations and exhibits protective e#ects in HIV-infected cells (Lamidey et al.,,**,; King and Robinson, +332). Furthermore, Pluymers et al. (,***) reported that D-chicoric acid has a stronger anti-hiv activity than L-chicoric acid. Compounds. and /, which were consistent with peaks / and 0 on the HPLC- PDA chromatogram (Fig.,), were determined as luteolin 1-O-b-D-glucopyranoside and luteolin 1-O-b-D-glucuronide, respectively. Luteolin is a type of flavonoid which has been reported to have strong antioxidative activity in the LDL oxidation system (Hirano et al.,,**+). Brown et al. (+332) demonstrated that the dose-dependent reduction of LDL oxidation of luteolin 1-O-b-D-glucopyranoside was less e#ective than that of luteolin. Shimoi et al. (+332) investigated the intestinal absorption of luteolin and luteolin 1-O-b-D-glucopyranoside in rats using HPLC.

7 14 They suggested that luteolin was converted to glucuronides while passing through the mucosa, and luteolin 1-O-b-D-glucopyranoside was absorbed after hydrolysis to luteolin. Furthermore, HPLC analysis revealed that both free luteolin and its monoglucuronide were present in human serum after ingestion of luteolin. The DPPH scavenging activity of compounds +,,, -,. and / were determined (Table -). All five compounds showed radical scavenging activities. The scavenging activity of the test compounds decreased in the following order: chicoric acid, luteolin 1-O-b-D-glucopyranoside, and luteolin 1-O-b-D-glucuronide ca#eic acid chlorogenic acid. Chicoric acid, luteolin 1-O-b-D-glucopyranoside, and luteolin 1-O-b-D-glucuronide were the major active compounds. Other active compounds may also be present in Hosobawadan extract, and are now under investigation. In summary, we determined the phenolic contents and the antioxidant activities of./ species of vegetables and found that eight Okinawan traditional vegetables had strong antioxidant activities as detected by various in vitro assay systems. Furthermore, five antioxidant compounds were isolated and identified from Hosobawadan, and their antioxidant activities were evaluated. The primary components were chicoric acid, luteolin 1-O-b-Dglucopyranoside and luteolin 1-O-b-D-glucuronide, which showed strong DPPH scavenging activities. As a result, it is suggested that Hosobawadan is an e$cient dietary source of chicoric acid and luteolin derivatives. Acknowledgements The authors thank Dr. Tojiro Tsushida for his editorial suggestions. We also thank Dr. Makoto Yoshimoto for providing the isochlorogenic acids. G. MAEDA et al. References Brown, J.E. and Rice-Evans, C.A. (+332). Luteolin-rich artichoke extract protects low density lipoprotein from oxidation in vitro, Free Rad. Res.,,3,,.1,//. Frankel, E.N., Kanner, J., German, J.B., Parks, E. and Kinsella, J.E. (+33-). Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine, Lancet, -.+,./../1. Frankel, E.N., Waterhouse, A.L. and Teissedre, P.L. (+33/). Principal phenolic phytochemicals in selected California wines and their antioxidant activity in inhibiting oxidation of human low-density lipoproteins, J. Agric. Food Chem.,.-, 23* 232. Furuta, S., Suda, I., Nishiba, Y. and Yamakawa, O. (+332). High tert-butylperoxyl radical scavenging activities of sweet potato cultivars with purple flesh, Food Sci. Technol. Int. Tokyo,., -- -/. Harrmann, K. (+323). Occurrence and content of hydroxycinnamic and hydroxybenzoic acid compounds in foods, Crit. Rev. Food Sci. Nutr.,,2, -+/ -.1. Ichiba, T. and Kyan, Y. (,***). Research report of Okinawa Industrial Technology Center, Gushikawa, pp.,, +,, (in Japanese). Hirano, R., Sasamoto, W., Matsumoto, A., Itakura, H., Igarashi, O. and Kondo, K. (,**+). Antioxidant ability of various flavonoids against DPPH and LDL oxidation, J Nutr. Sci. Vitaminol.,.1, -/1-0,. Kerry, N.L. and Abbey, M. (+331). Red wine and fractionated phenolic compounds prepared from red wine inhibit low density lipoprotein oxidation in vitro, Atherosclerosis, +-/, 3- +*,. King, P. J. and Robinson, W.E. Jr. (+332). Resistance to the antihuman immunodeficiency virus type + compound L-chicoric acid results from a single mutation at amino acid +.* of integrase, J. Virol., 1,, 2.,* 2.,.. Kondo, K., Matsumoto, A., Kurata, H., Tanahashi, H., Koda, H., Amachi, T. and Takura, H. (+33.). Inhibition of oxidation of low-density lipoprotein with red wine., Lancet, -.., ++/,. Lamidey, A.M., Fernon, L., Pouysegu, L., Delattre, C. and Quideau, S. (,**,). A convenient synthesis of the Echinacea-derived immunostimulator and HIV-+ integrase inhibitor ( )-(,R, -R)- chicoric acid, Helv. Chim. Acta, 2/,,-,2,--.. Lu, Y. and Foo, L.Y. (,***). Flavonoid and phenolic glycosides from Salvia o$cinalis, Phytochem., //,,0-,01. Morishita, H., (+32.). Chromatographic separation and identification of naturally occurring chlorogenic acids by + H nuclear magnetic resonance spectroscopy and mass spectrometry, J. Chromatogr., -+/,,/-,0*. Oki, T., Masuda, M., Kobayashi, M., Nishiba, Y., Furuta, S., Suda, I. and Sato, T. (,**+). Radical scavenging activity of fried chips made from purple-fleshed sweet potato, Nippon Shokuhin Kagaku Kogaku Kaishi,.2, 3,0 3-, (in Japanese). Oki T., Masuda M., Kobayashi M., Nishiba Y., Furuta S., Suda I. and Sato T. (,**,). Polymeric procyanidins as radical-scavenging components in red-hulled rice, J. Agric. Food Chem., /*, 1/,. 1/,3. Pluymers, W., Neamati, N., Pannecouque, C., Fikkert, V., Marchand, C., Burke, T.R. Jr., Pommier, Y., Schols, D., Clercq, E. D., Debyser, Z. and Mitvrouw, M. (,***). Viral entry as the primary target for the anti-hiv activity of chicoric acid and its tetra-acetyl esters, Mol. Pharmacol., /2, Renaud, S. and de Lorgeril, M. (+33,). Wine, alcohol, platelets, and the French paradox for coronary heart disease, Lancet, --3, +/,- +/,0. Shimoi, K., Okada, H., Furugori, M., Goda, T., Takase, S., Suzuki, M., Hara., Yamamoto, H. and Kinae, N. (+332). Intestinal absorption of luteolin and luteolin 1-O-b-D-glucopyranoside in rats and humans, FEBS Letters,.-2,,,*,,.. Syuyama, K., ed. (,***). Tyouzyu no youin., Kyushu University publication center, 2,,/3 (in Japanese). Tateyama, C., Honma, N., Namiki, K. and Uchiyama, T. (+331). Polyphenol content and antioxidant activity of various flower petals, Nippon Shokuhin Kagaku Kogaku Kaishi,..,,3*,33 (in Japanese). Takahata Y., Ohnishi-Kameyama M., Furuta S., Takahashi M. and Suda I. (,**+). Highly polymerized procyanidins in brown soybean seed coat with a high radical-scavenging activity, J. Agric. Food Chem.,.3, /2.- /2.1. Terao, J. (+33*). Lipid hydroperoxides : their occurrence and reaction in food and biological systems. Nippon Nogeikagaku Kaishi, 0., ,0 (in Japanese). Tsushida, T., Suzuki, M. and Kurogi, M. (+33.). Evaluation of antioxidant activity of vegetable extracts and determination of some antioxidants, Nippon Shokuhin Kagaku Kogaku Kaishi,.+, (in Japanese). Veit, M., Strack, D., Czygan, F.C., Wray, V. and Witte, L. (+33+). di-e-ca#eoyl-meso-tartaric acid in the barren sprouts of Equisetum Arvense, Phytochem., -*, /,1 /,3. Wang, M., Li, J., Rangarajan, M., Shao, Y., Lavoie, E. J., Huang, T. and Ho, C. (+332). Antioxidative phenolic compounds from sage (Salvia o$cinalis), J. Agric. Food Chem.,.0, Yoshimoto, M., Yahara, S., Okuno, S., Islam, M. S., Ishiguro, K. and Yamakawa, O. (,**,). Antimutagenicity of mono-, di-, and trica#eoylquinic acid derivatives isolated from sweet potato (Ipomoea batatas L.) leaf, Biosci. Biotechnol. Biochem., 00,,--0,-.+. Zou, J., Huang, Y., Chen, Q., Wei, E., Hsieh, T. and Wu, J.M. (+333). Resveratrol inhibits copper ion-induced and azo compoundinitiated oxidative modification of human low density lipoprotein, Biochem. Mol. Biol. Int.,.1, +*23 +*30.